Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

problem of membrane protein FPLC


  • Please log in to reply
1 reply to this topic

#1 handsome

handsome

    member

  • Members
  • Pip
  • 4 posts
0
Neutral

Posted 28 February 2010 - 08:42 PM

I purified my membrane protein by FPLC gel filtration column. Before the injection , I checked the OD280, which is around 1AU. then i took 0.5ml protein sample to run the FPLC. However, no sharp peak came out. I just got a broad and small peak with 35 mAu. which is much lower than that expected. (I purified the membrane protein with DM and the FPLC buffer : 150mM NaCl, 20mM HEPEs, 0.4g/100ml DM)

Is it that my protein has aggregated on the FPLC column?

I also try to increase salt concentration (300mM) of the FPLC buffer , then I got a sharp peak with OD 160 mAu and the retention time is some early than Void Volume. Is it normal ? anybody can give me some suggestion for the FPLC pufication condition?

#2 mdfenko

mdfenko

    an elder

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 2,805 posts
133
Excellent

Posted 02 March 2010 - 12:57 PM

which fplc column did you use?

recommended minimum ionic strength for fplc buffer (gel filtration) is 200mM. this prevents non-specific binding to the stationary phase.

if the peak came before the void then it was something released from the column prior to the injection. when you re-equilibrated with the higher salt buffer you probably did not completely wash the column before you injected (anything bound that was released will fractionate).
talent does what it can
genius does what it must
i do what i get paid to do




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.